Live, oral vaccine for protection against Shigella dysenteriae serotype 1

ABSTRACT

The invention relates to  Salmonella typhi  Ty21a comprising core-linked  Shigella dysenteriae  serotype 1 O-specific polysaccharide (O-Ps) and DNA encoding O antigen biosynthesis, said DNA selected from the group consisting of: a) the DNA sequence set out in any one of SEQ ID NOs: 1 and 2 and species homologs thereof; b) DNA encoding  Shigella dysenteriae  serotype 1 polypeptides encoded by any one of SEQ ID NOs: 1 and 2, and species homologs thereof; and c) DNA encoding a O antigen biosynthesis gene product that hybridizes under moderately stringent conditions to the DNA of (a) or (b); and related sequences, compositions of matter, vaccines, methods of using, and methods of making.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/145,104, filed Dec. 31, 2013, now U.S. Pat. No. 8,968,719, issuedMar. 3, 2015, which is a continuation of U.S. patent application Ser.No. 13/687,797, filed Nov. 28, 2012, now U.S. Pat. No. 8,790,635, issuedJul. 29, 2014, which is a continuation of U.S. patent application Ser.No. 13/285,614, filed Oct. 31, 2011, now U.S. Pat. No. 8,337,831, issuedDec. 25, 2012; which is a continuation of U.S. patent application Ser.No. 11/597,301, filed Sep. 21, 2007, now U.S. Pat. No. 8,071,113, issuedDec. 6, 2011; which is a national phase entry pursuant to 35 U.S.C. §371of International Patent Application No. PCT/US2005/018198, filed May 24,2005; which application claims the benefit of U.S. Provisional PatentApplication No. 60/609,494, filed Sep. 13, 2004, and U.S. ProvisionalPatent Application No. 60/574,279, filed May 24, 2004; the disclosuresof all of the foregoing applications are incorporated herein byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The instant application was made with government support; the governmenthas certain rights in this invention.

SEQUENCE LISTING

The Sequence Listing text file attached hereto, created Nov. 28, 2012,size 42 kilobytes, and filed herewith as file name“6137FDA3PUS12_SEQ_ST25.txt” is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Shigella cause millions of cases of dysentery (i.e., severe bloodydiarrhea) every year, which result in 660,000 deaths worldwide. Shigelladysenteriae serotype 1, one of about 40 serotypes of Shigella, causes amore severe disease with a much higher mortality rate than otherserotypes. There are no FDA-licensed vaccines available for protectionagainst Shigella, although a number of institutions are trying variousvaccine approaches. The fact that many isolates exhibit multipleantibiotic resistance complicates the management of dysenteryinfections. The development of an immunogenic composition againstShigella dysenteriae serotype 1 therefore represents a particularlyurgent objective.

SUMMARY OF THE INVENTION

The invention relates to Salmonella typhi Ty21a comprising core-linkedShigella dysenteriae serotype 1 O-specific polysaccharide (O-Ps) and DNAencoding O antigen biosynthesis, said DNA selected from the groupconsisting of:

-   -   (a) the DNA sequence set out in any one of SEQ ID NOs: 1 and 2        and species homologs thereof;    -   (b) DNA encoding Shigella dysenteriae serotype 1 polypeptides        encoded by any one of SEQ ID NOs: 1 and 2, and species homologs        thereof; and    -   (c) DNA encoding a O antigen biosynthesis gene product that        hybridizes under moderately stringent conditions to the DNA        of (a) or (b);        and related sequences, compositions of matter, vaccines, methods        of using, and methods of making.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. The genes necessary for biosynthesis of the Shigella dysenteriaeserotype 1O-antigen.

FIG. 2. E. coli (pGB2-Sd1) was found to express Shigella dysenteriaeserotype 1 O-antigen by both slide agglutination and immunoblot assaysusing Shigella dysenteriae serotype 1-specific antisera.

FIG. 3. Proposed sugar transferase requirements for synthesis of theShigella dysenteriae serotype 1 O-polysaccharide repeat unit. Rfe is aGlcNac transferase which adds GlcNAc to ACL (antigen carrier lipid/acyllipid carrier/undecaprenol phosphate); RfbR and RfbQ are Rhatransferases; Rfp is a galactosyl transferase (Mol. Microbial. 1995,18:209)

FIGS. 4A-4B. Expression analyses of LPS from various parental andplasmid-carrying strains. LPS was extracted from various strains asdescribed below and separated on SDS-PAGE gels by electrophoresis.Resulting silver-stained material (4A) and a Western immunoblot (4B)reacted with anti-Shigella dysenteriae serotype 1 antisera are shown. Inboth parts (4A) and (4B), molecular weight markers are shown in theleft-hand lane followed by extracted polysaccharide from E. colicarrying pGB2 (lane pGB2.E. coli), parent S. typhi Ty21a (lane TyphiTy21a), Ty21a carrying pGB2-Sd1 (lane Sd1.Ty21a), E. coli carryingpGB2-Sd1 (lane Sd1.E. coli), the parent Shigella dysenteriae serotype 1strain 1617 (lane Sd1 1617), or the rough Shigella dysenteriae serotype1 strain 60R (lane Sd1 60R).

SEQUENCE SUMMARY SEQ ID NO. Description 1 9297 bp. Sequence of rfb locusof Shigella dysenteriae serotype 1 strain 1617 2 1507 bp. rfp Sequencefrom Shigella dysenteriae serotype 1 strain 1617. 3 rfbB 4 rfbC 5 rfbA 6rfbD 7 rfbX 8 rfc 9 rfbR 10 rfbQ 11 orf9 12 rfp

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Shigella dysenteriae serotype 1 causes the most severe form ofshigellosis, often associated with hemolytic uremic syndrome inchildren, especially in developing countries. Due to the high level ofShiga toxin production and associated high morbidity/mortality, thisorganism is classified as a Category B bioterrorist threat agent. Thelipopolysaccharide of Shigella dysenteriae serotype 1 is essential forvirulence, and there is indirect evidence that antibodies against thisO-specific polysaccharide (O-Ps) are protective to the host. Thus, thereis considerable interest in the development of an O-Ps-based vaccine toprotect against Shigella dysenteriae serotype 1. Previous studies showedthat the determinants for the production of O antigen lipopolysaccharidein Shigella dysenteriae serotype 1 are distributed on the chromosome(i.e., rfb/rfc genes) and on a small 9-kb plasmid (i.e., rfp gene). Thecurrent studies were aimed at cloning the Rfb/Rfc region from strain1617 to define all essential genes and develop a biosynthetic pathwayfor O-Ps biosynthesis. The plasmid-carried gene (i.e., the rfp-encodedgalactosyl transferase) was also cloned from strain 1617; its 1.6 kbsequence was found to be >99% homologous to rfp previously analyzed froma different Shigella dysenteriae serotype 1 strain. Additionally, thechromosomal Rfb/Rfc region of 9 kb was cloned and sequenced, and foundto contain 9 ORFs. Preliminary analysis suggests that all 9 ORFs plusrfp are necessary for serotype 1 LPS biosynthesis. We anticipate thatthe use of these characterized O-Ps genes in a live, attenuatedSalmonella delivery system will lead to a safe, oral vaccine forprotection against this severe form of shigellosis.

Introduction

Shigella spp. are the predominant cause of acute bloody diarrhea(dysentery) worldwide, and cause 660,000 deaths globally each year dueto shigellosis. Infection with Shigella dysenteriae serotype 1 strainscauses a more severe illness with higher mortality than with otherShigellae, particularly in young children and the elderly.

Protective immunity against shigellosis appears to be serotype-specificand protection correlates with the stimulation of immunity against theO-specific surface lipopolysaccharide.

The genes necessary for O-Ps synthesis in Shigella dysenteriae serotype1 lie on a 9-kb small plasmid (i.e., the rfp gene) and on the chromosome(i.e., rfb cluster). A recombinant plasmid containing the essentialShigella dysenteriae serotype 1 O-antigen biosynthetic genes waspreviously constructed and introduced into E. coli or attenuatedSalmonella spp. This plasmid construct was reported to be unstable whenthe strains were cultivated without selective pressure, and animalimmunization resulted in less than 50% protection (Klee, S. R. et al.1997 J Bacteriol 179:2421-2425).

The current studies were aimed at cloning the essential O-Psbiosynthetic machinery of Shigella dysenteriae serotype 1, deletingunnecessary adjacent sequences, and completing the DNA sequence analysisof the entire biosynthetic region to define a minimal essential set ofgenes.

Materials and Methods

1. Shigella dysenteriae serotype 1 strain 1617 was obtained from theculture collection of S. B. Formal, Walter Reed Army Institute ofResearch (WRAIR). The strain was originally isolated from an outbreak ofepidemic Shiga bacillus dysentery in Guatemala, Central America, in 1968(Mendizabal-Morris, C A. et al. 1971 J Trop Med Hyg 20:927-933). Plasmidand chromosomal DNA used in this study was prepared from this strain.

2. The plasmid rfp region and its cognate promoter and a ˜9.5 kb rfblocus were first cloned into the pCR 2.1-TOPO vector separately. Theinsert DNA was confirmed by DNA sequence analysis, and then transferredinto the low copy plasmid pGB2 for genetic stabilization.

3. DNA sequence analysis and BLAST homology searches were employed tocharacterize the essential biosynthetic gene region.

4. The parent Shigella dysenteriae serotype 1 strain 1617 andrecombinant E. coli strains expressing the Shigella dysenteriae serotype1 O-Ps were analyzed for expression by agglutination and immunoblotassays with specific anti-Shigella dysenteriae serotype 1 LPS antisera(Difco, Detroit).

TABLE 1 Summary of Shigella dysenteriae serotype 1 O-Ps ORFs ORF Genename Location Proposed function 1 rfbB 756-1841 dTDP-D-glucose 4,6dehydratase 2 rfbC 1841-2740 dTDP-4-dehydrorhamnose reductase 3 rfbA2798-3676 Glucose-I-phosphate thymilytransferase 4 rfbD 3679-4236dTDP-4-dehydrorhamnose 3,5-epimerase 5 rfbX 4233-5423 O-Ag transporter 6rfc 5420-6562 O-Ag polymerase 7 rfbR 6555-7403 dDTP-rhamnosyltransferase 8 rfbQ 7428-8339 Rhamnosyltransferase 9 orf9 8349-8783Galactosyltransferase (?) 10 rfp 1134 bp (on Galactosyltransferase smallplasmid

SUMMARY

Referring to Table 1 and FIGS. 1-3:

1. The O-Ps biosynthetic determinants from Shigella dysenteriae serotype1 strain 1617 were cloned from both the chromosome (i.e., rfb locus) anda small 9 kb plasmid (i.e., the rfp gene).

2. The separate rfb locus (GenBank accession: AY585348) and rfP region(GenBank accession: AY763519) covering ˜11 kb total DNA were sequencedentirely and revealed a total of 10 ORFs apparently necessary for O-Psbiosynthesis.

3. A low copy pGB2 vector containing both the rfb and rfp loci in tandemlinkage was constructed (i.e., pGB2-Sd1) and found to express Shigelladysenteriae serotype 1 O-Ps antigen.

4. Requirements for sugar linkage in the final O-Ps structure ofShigella dysenteriae serotype 1 are proposed.

5. We anticipate that use of this cloned antigen locus in a live,attenuated Salmonella delivery system will lead to a safe, oral vaccinefor protection against this severe form of shigellosis.

Part 1

In one embodiment, the invention comprises a prokaryotic microorganism.Preferably, the prokaryotic microorganism is an attenuated strain ofSalmonella. However, alternatively other prokaryotic microorganisms suchas attenuated strains of Escherichia coli, Shigella, Yersinia,Lactobacillus, Mycobacteria, Listeria or Vibrio could be used. Examplesof suitable strains of microorganisms include Salmonella typhimurium,Salmonella typhi, Salmonella dublin, Salmonella enteritidis, Escherichiacoli, Shigella flexneri, Shigella sonnet, Vibrio cholera, andMycobacterium bovis (BCG).

In a preferred embodiment the prokaryotic microorganism is Salmonellatyphi Ty21a. Vivotif® Typhoid Vaccine Live Oral Ty21a is a liveattenuated vaccine for oral administration only. The vaccine containsthe attenuated strain Salmonella typhi Ty21a. (Germanier et al. 1975 JInfect. Dis. 131:553-558). It is manufactured by Bema Biotech Ltd.Berne, Switzerland. Salmonella typhi Ty21a is also described in U.S.Pat. No. 3,856,935.

As mentioned above, the attenuated strain of the prokaryoticmicroorganism is transformed with a nucleic acid encoding one or moreO-Ps genes. The inventors found for the first time that, when thisnucleic acid is expressed in the microorganisms, core-linked O-Ps LPSare generated.

In a further aspect, the present invention provides a compositioncomprising one or more of above attenuated prokaryotic microorganisms,optionally in combination with a pharmaceutically or physiologicallyacceptable carrier. Preferably, the composition is a vaccine, especiallya vaccine for mucosal immunization, e.g., for administration via theoral, rectal, nasal, vaginal or genital routes. Advantageously, forprophylactic vaccination, the composition comprises one or more strainsof Salmonella expressing a plurality of different O-Ps genes.

In a further aspect, the present invention provides an attenuated strainof a prokaryotic microorganism described above for use as a medicament,especially as a vaccine.

In a further aspect, the present invention provides the use of anattenuated strain of a prokaryotic microorganism transformed withnucleic acid encoding enzymes for O-Ps synthesis, wherein the O-Ps areproduced in the microorganism, in the preparation of a medicament forthe prophylactic or therapeutic treatment of bacterial infection.

Generally, the microorganisms or O-Ps according to the present inventionare provided in an isolated and/or purified form, i.e., substantiallypure. This may include being in a composition where it represents atleast about 90% active ingredient, more preferably at least about 95%,more preferably at least about 98%. Such a composition may, however,include inert carrier materials or other pharmaceutically andphysiologically acceptable excipients. A composition according to thepresent invention may include in addition to the microorganisms or O-Psas disclosed, one or more other active ingredients for therapeutic orprophylactic use, such as an adjuvant.

The compositions of the present invention are preferably given to anindividual in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be, althoughprophylaxis may be considered therapy), this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g., decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may include, in additionto active ingredient, a pharmaceutically or physiologically acceptableexcipient, carrier, buffer, stabilizer or other materials well known tothose skilled in the art. Such materials should be non-toxic and shouldnot interfere with the efficacy of the active ingredient. The precisenature of the carrier or other material will depend on the route ofadministration.

Examples of techniques and protocols mentioned above can be found inRemington's Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.

The invention further relates to the identification and sequencing of 9ORFs in the rfb locus (GenBank accession number: AY585348) and an ORF inthe rfb locus (GenBank accession number: AY763519). These genes may bepresent in whole or in part in the vaccine strains described herein.

Accordingly, the present invention relates to vaccine strains furthercharacterized by the presence of heterologous genes or a set ofheterologous genes coding for O-Ps.

In a preferred embodiment of the vaccine strains, the heterologousgene(s) is (are) present either on a plasmid vector or stably integratedinto the chromosome of said strain at a defined integration site whichis to be nonessential for inducing a protective immune response by thecarrier strain.

In a preferred embodiment, the heterologous genes of the invention,including all 9 ORFs from the rfb locus and the ORF from rfp, arepresent on a plasmid derived from pGB2 (Churchward et al. 1984 Gene31:165-171). In another embodiment, the ninth ORF from rfb is notpresent, because it is not essential for O-Ps biosynthesis.

The ORFs may be under the control of the cognate promoter or othernon-cognate promoters. The rfb genes may be separated and present onseparate polynucleotide molecules under the control of differentpromoters, or on the same polynucleotide molecule in any order.

Alternatively, the above vaccine strains contain the rfbB, rfbC, andrfbA and/or any additional gene(s) necessary for the synthesis ofcomplete core-linked O-antigen LPS which are integrated in tandem into asingle chromosomal site or independently integrated into individualsites, or cloned into a plasmid or plasmids.

Such vaccine strains allow expression of heterologous O-Ps which iscovalently coupled to a heterologous LPS core region, which, preferably,exhibits a degree of polymerization essentially indistinguishable fromthat of native LPS produced by the enteric pathogen. Such vaccinestrains can, if desired, modified in such a way that they are deficientin the synthesis of homologous LPS core.

The invention also relates to a live vaccine comprising the abovevaccine strain and optionally a pharmaceutically or physiologicallyacceptable carrier and/or a buffer for neutralizing gastric acidityand/or a system for delivering said vaccine in a viable state to theintestinal tract.

Said vaccine comprises an immuno-protective or -therapeutic andnon-toxic amount of said vaccine strain. Suitable amounts can bedetermined by the person skilled in the art and are typically 10⁷ to 10⁹bacteria.

Pharmaceutically and physiologically acceptable carriers, suitableneutralizing buffers, and suitable delivering systems can be selected bythe person skilled in the art.

In a preferred embodiment said live vaccine is used for immunizationagainst gram-negative enteric pathogens.

The mode of administration of the vaccines of the present invention maybe any suitable route which delivers an immunoprotective orimmunotherapeutic amount of the vaccine to the subject. However, thevaccine is preferably administered orally or intranasally.

The invention also relates to the use of the above vaccine strains forthe preparation of a live vaccine for immunization against gram-negativeenteric pathogens. For such use the vaccine strains are combined withthe carriers, buffers and/or delivery systems described above.

The invention also provides polypeptides and correspondingpolynucleotides required for synthesis of core linked O-specificpolysaccharide. The invention includes both naturally occurring andunnaturally occurring polynucleotides and polypeptide products thereof.Naturally occurring O antigen biosynthesis products include distinctgene and polypeptide species as well as corresponding species homologsexpressed in organisms other than Shigella dysenteriae serotype 1strains. Non-naturally occurring O antigen biosynthesis products includevariants of the naturally occurring products such as analogs and Oantigen biosynthesis products which include covalent modifications. In apreferred embodiment, the invention provides O antigen biosynthesispolynucleotides comprising the sequences set forth in SEQ ID NOs: 1 and2 and species homologs thereof, and polypeptides having amino acidssequences encoded by the polynucleotides.

The present invention provides novel purified and isolated Shigelladysenteriae serotype 1 polynucleotides (e.g., DNA sequences and RNAtranscripts, both sense and complementary antisense strands) encodingthe bacterial O antigen biosynthesis gene products. DNA sequences of theinvention include genomic and cDNA sequences as well as wholly orpartially chemically synthesized DNA sequences. Genomic DNA of theinvention comprises the protein coding region for a polypeptide of theinvention and includes variants that may be found in other bacterialstrains of the same species. “Synthesized,” as used herein and isunderstood in the art, refers to purely chemical, as opposed toenzymatic, methods for producing polynucleotides. “Wholly” synthesizedDNA sequences are therefore produced entirely by chemical means, and“partially” synthesized DNAs embrace those wherein only portions of theresulting DNA were produced by chemical means. Preferred DNA sequencesencoding Shigella dysenteriae serotype 1 O antigen biosynthesis geneproducts are set out in SEQ ID NOs: 1 and 2, and species homologsthereof.

The worker of skill in the art will readily appreciate that thepreferred DNA of the invention comprises a double-stranded molecule, forexample, molecules having the sequences set forth in SEQ ID NOs: 1 and 2and species homologs thereof, along with the complementary molecule (the“non-coding strand” or “complement”) having a sequence deducible fromthe sequence of SEQ ID NOs: 1 and 2, according to Watson-Crickbasepairing rules for DNA. Also preferred are polynucleotides encodingthe gene products encoded by any one of the polynucleotides set out inSEQ ID NOs: 1 and 2 and species homologs thereof.

The invention also embraces DNA sequences encoding bacterial geneproducts which hybridize under moderately to highly stringent conditionsto the non-coding strand, or complement, of any one of thepolynucleotides set out in SEQ ID NOs: 1 and 2, and species homologsthereof. DNA sequences encoding O antigen biosynthesis polypeptideswhich would hybridize thereto but for the degeneracy of the genetic codeare contemplated by the invention. Exemplary high stringency conditionsinclude a final wash in buffer comprising 0.2×SSC/0.1% SDS, at 65° C. to75° C., while exemplary moderate stringency conditions include a finalwash in buffer comprising 2×SSC/0.1% SDS, at 35° C. to 45° C. It isunderstood in the art that conditions of equivalent stringency can beachieved through variation of temperature and buffer, or saltconcentration as described in Ausubel, et al. (eds.), Short Protocols inMolecular Biology, John Wiley & Sons (1994), pp. 6.0.3 to 6.4.10.Modifications in hybridization conditions can be empirically determinedor precisely calculated based on the length and the percentage ofguanosine-cytosine (GC) base pairing of the probe. The hybridizationconditions can be calculated as described in Sambrook, et al., (eds.),Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress: Cold Spring Harbor, N.Y. (1989), pp. 9.47 to 9.51.

Autonomously replicating recombinant expression constructions such asplasmid and viral DNA vectors incorporating O antigen biosynthesis genesequences are also provided. Expression constructs wherein O antigenbiosynthesis polypeptide-encoding polynucleotides are operatively linkedto an endogenous or exogenous expression control DNA sequence and atranscription terminator are also provided. The O antigen biosynthesisgenes may be cloned by PCR, using Shigella dysenteriae serotype 1genomic DNA as the template. For ease of inserting the gene intoexpression vectors, PCR primers are chosen so that the PCR-amplifiedgene has a restriction enzyme site at the 5′ end preceding theinitiation codon ATG, and a restriction enzyme site at the 3′ end afterthe termination codon TAG, TGA or TAA. If desirable, the codons in thegene are changed, without changing the amino acids, according to E. colicodon preference described by Grosjean and Fiers, 1982 Gene 18: 199-209;and Konigsberg and Godson, 1983 PNAS USA 80:687-691. Optimization ofcodon usage may lead to an increase in the expression of the geneproduct when produced in E. coli. If the gene product is to be producedextracellularly, either in the periplasm of E. coli or other bacteria,or into the cell culture medium, the gene is cloned without itsinitiation codon and placed into an expression vector behind a signalsequence.

According to another aspect of the invention, host cells are provided,including procaryotic and eukaryotic cells, either stably or transientlytransformed, transfected, or electroporated with polynucleotidesequences of the invention in a manner which permits expression of Oantigen biosynthesis polypeptides of the invention. Expression systemsof the invention include bacterial, yeast, fungal, viral, invertebrate,and mammalian cells systems. Host cells of the invention are a valuablesource of immunogen for development of anti-bodies specificallyimmunoreactive with the O antigen biosynthesis gene product. Host cellsof the invention are conspicuously useful in methods for large scaleproduction of O antigen biosynthesis polypeptides wherein the cells aregrown in a suitable culture medium and the desired polypeptide productsare isolated from the cells or from the medium in which the cells aregrown by, for example, immunoaffinity purification or any of themultitude of purification techniques well known and routinely practicedin the art. Any suitable host cell may be used for expression of thegene product, such as E. coli, other bacteria, including P. multocida,Bacillus and S. aureus, yeast, including Pichia pastoris andSaccharomyces cerevisiae, insect cells, or mammalian cells, includingCHO cells, utilizing suitable vectors known in the art. Proteins may beproduced directly or fused to a peptide or polypeptide, and eitherintracellularly or extracellularly by secretion into the periplasmicspace of a bacterial cell or into the cell culture medium. Secretion ofa protein requires a signal peptide (also known as pre-sequence); anumber of signal sequences from prokaryotes and eukaryotes are known tofunction for the secretion of recombinant proteins. During the proteinsecretion process, the signal peptide is removed by signal peptidase toyield the mature protein.

To simplify the protein purification process, a purification tag may beadded either at the 5′ or 3′ end of the gene coding sequence. Commonlyused purification tags include a stretch of six histidine residues (U.S.Pat. Nos. 5,284,933 and 5,310,663), a streptavidin affinity tagdescribed by Schmidt and Skerra, (1993 Protein Engineering 6:109-122), aFLAG peptide (Hopp et al. 1988 Biotechnology 6:1205-1210), glutathione5-transferase (Smith and Johnson, 1988 Gene 67:31-40), and thioredoxin(LaVallie et at. 1993 Bio/Technology 11:187-193). To remove thesepeptide or polypeptides, a proteolytic cleavage recognition site may beinserted at the fusion junction. Commonly used proteases are factor Xa,thrombin, and enterokinase.

The invention also provides purified and isolated Shigella dysenteriaeserotype 1 O antigen biosynthesis polypeptides encoded by apolynucleotide of the invention. Presently preferred are polypeptidescomprising the amino acid sequences encoded by any one of thepolynucleotides set out in SEQ ID NOs: 1 and 2, and species homologsthereof. The invention embraces O antigen biosynthesis polypeptidesencoded by a DNA selected from the group consisting of:

a) the DNA sequence set out in any one of SEQ ID NOs: 1 and 2 andspecies homologs thereof;

b) DNA molecules encoding Shigella dysenteriae serotype 1 polypeptidesencoded by any one of SEQ ID NOs: 1 and 2, and species homologs thereof;and

c) a DNA molecule encoding a O antigen biosynthesis gene product thathybridizes under moderately stringent conditions to the DNA of (a) or(b).

The invention also embraces polypeptides that have at least about 99%,at least about 95%, at least about 90%, at least about 85%, at leastabout 80%, at least about 75%, at least about 70%, at least about 65%,at least about 60%, at least about 55%, and at least about 50% identityand/or homology to the preferred polypeptides of the invention. Percentamino acid sequence “identity” with respect to the preferredpolypeptides of the invention is defined herein as the percentage ofamino acid residues in the candidate sequence that are identical withthe residues in the O antigen biosynthesis gene product sequence afteraligning both sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Percentsequence “homology” with respect to the preferred polypeptides of theinvention is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical with the residues in one ofthe O antigen biosynthesis polypeptide sequences after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and also considering any conservativesubstitutions as part of the sequence identity. Conservativesubstitutions can be defined as set out in Tables A and B.

TABLE A Conservative Substitutions I SIDE CHAIN CHARACTERISTIC AMINOACID Aliphatic Non-polar G, A, P I, L, V Polar-uncharged C, S, T, M N, QPolar-charged D, E K, R Aromatic H, F, W, Y Other N, Q, D, E

Polypeptides of the invention may be isolated from natural bacterialcell sources or may be chemically synthesized, but are preferablyproduced by recombinant procedures involving host cells of theinvention. O antigen biosynthesis gene products of the invention may befull length polypeptides, biologically active fragments, or variantsthereof which retain specific biological or immunological activity.Variants may comprise O antigen biosynthesis polypeptide analogs whereinone or more of the specified (i.e., naturally encoded) amino acids isdeleted or replaced or wherein one or more non-specified amino acids areadded: (1) without loss of one or more of the biological activities orimmunological characteristics specific for the O antigen biosynthesisgene product; or (2) with specific disablement of a particularbiological activity of the O antigen biosynthesis gene product. Deletionvariants contemplated also include fragments lacking portions of thepolypeptide not essential for biological activity, and insertionvariants include fusion polypeptides in which the wild-type polypeptideor fragment thereof have been fused to another polypeptide.

Variant O antigen biosynthesis polypeptides include those whereinconservative substitutions have been introduced by modification ofpolynucleotides encoding polypeptides of the invention. Conservativesubstitutions are recognized in the art to classify amino acidsaccording to their related physical properties and can be defined as setout in Table A (from W097/09433, page 10). Alternatively, conservativeamino acids can be grouped as defined in Lehninger, (Biochemistry,Second Edition; W.H. Freeman & Co. 1975, pp.71-77) as set out in TableB.

TABLE B Conservative Substitutions II SIDE CHAIN CHARACTERISTIC AMINOACID Non-polar (hydrophobic) A. Aliphatic: A, L, I, V, P B. Aromatic: F,W C. Sulfur-containing: M D. Borderline: G Uncharged-polar A. Hydroxyl:S, T, Y B. Amides: N, Q C. Sulfhydryl: C D. Borderline: G PositivelyCharged (Basic): K, R, H Negatively Charged (Acidic): D, E

Variant O antigen biosynthesis products of the invention include matureO antigen biosynthesis gene products, i.e., wherein leader or signalsequences are removed, having additional amino terminal residues. Oantigen biosynthesis gene products having an additional methionineresidue at position −1 are contemplated, as are O antigen biosynthesisproducts having additional methionine and lysine residues at positions−2 and −1. Variants of these types are particularly useful forrecombinant protein production in bacterial cell types. Variants of theinvention also include gene products wherein amino terminal sequencesderived from other proteins have been introduced, as well as variantscomprising amino terminal sequences that are not found in naturallyoccurring proteins.

The invention also embraces variant polypeptides having additional aminoacid residues which result from use of specific expression systems. Forexample, use of commercially available vectors that express a desiredpolypeptide as fusion protein with glutathione-S-transferase (GST)provide the desired polypeptide having an additional glycine residue atposition −1 following cleavage of the GST component from the desiredpolypeptide. Variants which result from expression using other vectorsystems are also contemplated.

Also comprehended by the present invention are antibodies (e.g.,monoclonal and polyclonal antibodies, single chain antibodies, chimericantibodies, humanized, human, and CDR-grafted antibodies, includingcompounds which include CDR sequences which specifically recognize apolypeptide of the invention) and other binding proteins specific for Oantigen biosynthesis gene products or fragments thereof. The term“specific for” indicates that the variable regions of the antibodies ofthe invention recognize and bind a O antigen biosynthesis polypeptideexclusively (i.e., are able to distinguish a single O antigenbiosynthesis polypeptides from related O antigen biosynthesispolypeptides despite sequence identity, homology, or similarity found inthe family of polypeptides), but may also interact with other proteins(for example, S. aureus protein A or other antibodies in ELISAtechniques) through interactions with sequences outside the variableregion of the antibodies, and in particular, in the constant region ofthe molecule. Screening assays to determine binding specificity of anantibody of the invention are well known and routinely practiced in theart. For a comprehensive discussion of such assays, see Harlow et al.(eds.), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognizeand bind fragments of the O antigen biosynthesis polypeptides of theinvention are also contemplated, provided that the antibodies are firstand foremost specific for, as defined above, a O antigen biosynthesispolypeptide of the invention from which the fragment was derived.

Part II Molecular Characterization of Genes for Shigella DysenteriaeSerotype 1 O-Antigen and Expression in a Live Salmonella Vaccine Vector

Abstract

Shigella dysenteriae serotype 1, a bioterrorist threat agent, causes themost severe form of shigellosis and is typically associated with highmortality rates, especially in developing countries. This severe diseaseis due largely to Shiga-toxin-induced hemorrhagic colitis, plushemolytic uremic syndrome in children. The lipopolysaccharide ofShigella dysenteriae serotype 1 is essential for virulence, and there issubstantial evidence that antibodies against Shigella O-specificpolysaccharide (O-Ps) are protective to the host. Thus, there isconsiderable interest in the development of an O-Ps-based vaccine toprotect against Shigella dysenteriae serotype 1. Previous studies haveshown that the genetic determinants for the production of O-Ps antigenin Shigella dysenteriae serotype 1 are uniquely distributed on thechromosome (i.e., rfb genes) and on a small 9 kb plasmid (i.e., the rfpgene). In the current studies, the multi-ORF rfb gene cluster and therfp gene with their cognate promoter regions have been amplified by PCRfrom Shigella dysenteriae serotype 1 strain 1617. The two interrelatedbiosynthetic gene loci were then cloned and sequenced. Sequencingstudies revealed 9 ORFs located in the amplified 9.2 kb rfb region.Further deletion studies showed that only eight ORFs in the rfb regionare necessary, together with rfp, for Shigella dysenteriae serotype 1O-Ps biosynthesis. A linked rfb-rfp gene region cassette was constructedand cloned into the low copy plasmid pGB2, resulting in the recombinantplasmid designated pGB2-Sd1. When introduced by transformation intoeither Salmonella enterica serovar Typhi Ty21a or E. coli K-12, pGB2-Sd1directed the formation of surface-expressed, core-linked Shigelladysenteriae serotype 1 O-specific lipopolysaccharide. Silver stain andWestern immunoblotting analyses showed that the distribution of O repeatunits in S. typhi or E. coli K-12 was similar when compared with thepattern observed for the wild type strain 1617 of Shigella dysenteriaeserotype 1. In addition, a proposed biopathway, based upon ORF sequencehomologies to known genes, was developed. We anticipate that theinsertion of these jointly-cloned, O-Ps biosynthetic loci in a live,bacterial vaccine delivery system, such as attenuated S. typhi, willproduce a safe, oral vaccine for protection against this severe form ofshigellosis.

Introduction

Bacillary dysentery is a severe inflammation of the colon causedclassically by the entero-invasive bacterial genus Shigella. Theestimated number of bacillary dysentery infections worldwide is over 200million annually, with more than 650,000 associated deaths globally eachyear (Kotloff, K. L. et al. 1999 Bull World Health Organ 77:651-66).Shigellosis, especially in developing countries, is predominantly adisease of childhood. More than half of the cases occur in children lessthan 5 years of age, Shigellosis is highly transmissible due to the verylow infective dose of Shigella (i.e., <100 bacteria) and bacterialspread via the fecal-oral route (DuPont, H. L. et al. 1989 J Infect Dis159:1126-1128). Shigella dysenteriae serotype 1 (Shiga 1) is the primarycausative agent of epidemic outbreaks of severe bacillary dysenterywhich is associated with increased mortality. Due to the presence ofhigh levels of Shiga toxin produced by Shigella dysenteriae serotype 1strains, infections are more severe than those caused by other Shigellaspps. and are often characterized by serious complications (e.g.,hemolytic-uremic syndrome, hemorrhagic colitis, sepsis, and purpura)(Levine, M. M. 1982 Med Clin North Am 66:623-638). In addition, theemergence of strains resistant to multiple antibiotics makes therapeutictreatment difficult, particularly in developing countries, andemphasizes the need for vaccines in disease control. For these reasons,the World Health Organization (WHO) has given high priority to thedevelopment of a protective vaccine against Shigella dysenteriaeserotype 1 (Oberhelman, R. A. et al. 1991 Bull World Health Organ69:667-676). The increased concern for the potential use of this food-and water-borne pathogen of high morbidity and mortality as abioterrorist agent has recently amplified the interest in developing ananti-Shiga 1 vaccine.

Protective immunity against shigellosis is serotype-specific andcorrelates with stimulation of both systemic and local intestinalimmunity against the O-specific surface lipopolysaccharide (LPS) (Viret,J. F. et al. 1994 Biologicals 22:361-372; Winsor, D. K. et al. 1988 JInfect Dis 158:1108-1112). Genes for Shigella dysenteriae serotype 1 Oantigen biosynthesis are uniquely located in two unlinked gene clusters;one gene, rfp is located unusually on a 9 kb multicopy plasmid(Watanabe, H. et al. 1984 Infect Immun 43:391-396), and the remainingbiosynthetic genes are clustered, as usual, in the rfb chromosomal locus(Hale, T. L. et al. 1984 Infect Immun 46:470-5; Sturm, S. et al. 1986Microb Pathog 1:289-297). The O-Ps of Shigella dysenteriae serotype 1consists of the repeating tetrasaccharide unit: -3)-alpha-L-Rhap(1-3)-alpha-L-Rhap (1-2)-alpha-D-Galp (1-3)-alpha D-GlcNAcp (1-coreoligosaccharide. (Dmitriev, B. A. et al. 1976 Eur J Biochem 66:559-566;Falt, I. C. et al. 1996 Microb Pathog 20:11-30.)

The availability of a safe Salmonella typhi live, oral vaccine strainsince late 1970's stimulated new research efforts with the goals ofexpressing protective antigens (e.g., Shigella O-Ps) in an S. typhicarrier that could be used as a hybrid vaccine (e.g., to protect againstbacillary dysentery or other diseases) (Formal, S. B. et al. 1981 InfectImmun 34:746-50). In this initial study, the S. typhi Ty21a strain wasemployed as a delivery vector for expression of the form 1 O-Ps antigenof S. sonnei. However, the protection in volunteers provided byimmunizing with this hybrid vaccine strain varied (Herrington, D. A. etal. 1990 Vaccine 8:353-357), presumably due to spontaneous, highfrequency deletion of the form 1 gene region from a very large 300 kbcointegrate plasmid in vaccine strain 5076-IC (Hartman, A. B. et al.1991 J Clin Microbiol 29:27-32). In more recent studies, we haveconstructed a refined S. sonnei-Ty21a bivalent vaccine strain by usingthe defined O antigen gene cluster cloned into a genetically stable lowcopy plasmid. This refined hybrid vaccine strain showed highly stableexpression of form 1 antigen and following immunization it protectedmice against a stringent challenge with virulent S. sonnei (Xu, D. Q. etal. 2002 Infect Immun 70:4414-23).

In a similar vaccine development approach, the rfp gene and genes of therfb cluster of Shigella dysenteriae serotype 1 were introduced togetherinto attenuated strains of S. typhimurium (Falt, I. C. et al. 1996Microb Pathog 20:11-30), S. typhi (Mills, S. D. et al. 1988 Vaccine6:11622), or Shigella flexneri (Klee, S. R. et al. 1997 Infect Immun65:2112-2118) to create vaccine candidates for protection from thisShigella serotype. However, the Shigella dysenteriae serotype 1 O-Psantigen was expressed as core-linked in Shigella and in S. typhimurium(Falt, I. C. et al. 1996 Microb Pathog 20: 11-30), but was reportedlynot core-linked in S. typhi (Mills, S. D. et al. 1988 Vaccine 6:116-22).In the current studies, the Shigella dysenteriae serotype 1 O antigengene loci were cloned, sequenced completely and analyzed. Putative genesinvolved in synthesis of the tetrasaccharide O-repeating unit includingL-Rhap, L-Rhap, D-Galp, and D-GlcNAcp, as well as genes for O-unitprocessing and polymerization were identified. The four Rfb genesinvolved in rhamnose biosynthesis in Shigella dysenteriae serotype 1were found to be identical to those of E. coli 026, indicating commonancestry. In contrast to a previous report, analyses for the expressionof LPS in S. typhi Ty21a carrying the Shigella dysenteriae serotype 1O-antigen encoding rfb-rfp genes showed that the O-antigen repeat unitsare linked to the Salmonella typhi core and are envisioned asstimulating protection in mice against challenge with virulent Shigelladysenteriae serotype 1.

Materials and Methods

Bacterial strains, plasmids and growth conditions. The bacterial strainsand plasmids utilized are described in Table 2. The wild type parentShigella dysenteriae serotype 1 strain 1617 was obtained from S. B.Formal, Walter Reed Army Institute of Research (WRAIR) (Neill, R. J. etal. 1988 J Infect Dis 158:737-741). The strain was originally isolatedfrom an outbreak of epidemic Shiga bacillus dysentery in Guatemala,Central America, in 1968 or early 1969 (Mendizabal-Morris, C A. et al.1971 J Trop Med Hyg 20:927-933). The isolated strain 1617 waslyophilized and has been stored in sealed glass ampules. This strain issensitive to ampicillin, spectinomycin, streptomycin, tetracycline,chloramphenicol, and kanamycin. Strain 1617 was used to obtain theO-antigen biosynthetic genes and as a positive control for LPSexpression analyses. Studies of plasmid-based Shigella dysenteriaeserotype 1 LPS expression were performed in Escherichia coli DH5α, andSalmonella enterica serovar Typhi strain Ty21a. Shigella dysenteriaeserotype 1 strain 60R (rough strain, Spc^(r) which has lost the smallplasmid carrying rfp) was used as an LPS-negative control.

TABLE 2 Bacterial strains and plasmids Reference or Strain or plasmidGenotype or description source E. coli DH5α supE44 hsdR17 recA1 endA1gyrA96 thi-1 relA1 Sambrook, J. et al. (E. coli K12-origined) 1989Molecular cloning: a laboratory manual, 2^(nd) ed. Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. E. coli XL 1-blue supE44hsdR17 rec A1 endA1 gyrA96 thi-1 relA1 Sambrook, J. et al. lac[F′ proABlacIq ZM15Tn10 (Tet^(r))] (K12- 1989 (supra) DH5α-origined) E. coliTOP10F′ F′ {lacI^(q) Tn10 (Tet^(r)} mcrA Δ(mmr-hsdRMS- Invitrogen mcrBC)φ80lacZ ΔM15 Δ lacX74 recA1araD139 Δ(ara-leu)7697 galU galK rpsL(Str^(r)) endA1 nupG Salmonella enterica serovar Typhi Ty21a galE ilvDviaB (Vi) h2S Germanier, R. and E. Furer 1975 J Infect Dis 131: 553-558Shigella dysenteriae 1617, virulent S. Formal (Neill, R. J. serotype 1et al. 1988 J Infect Dis 158: 737-741) Shigella dysenteriae rough LPSmutant missing rfp plasmid S. Formal (Neill, R. J. serotype 1 60R et al.1988 J Infect Dis 158: 737-741) Plasmid pGB2 pSC101 derivative, low copyplasmid; Sm^(r), Spc^(r) Churchward, G. et al. 1984 Gene 31: 165-171pCR2.1-TOPO PCR TA cloning vector, pUC origin, Amp^(r) Kan^(r)Invitrogen pXK-Tp pCR2.1-TOPO containing rfp gene of strain 1617, thisstudy Amp^(r) Kan^(r) pXK-Bp56 pGB2 containing rfp gene of strain 1617,Spc^(r) this study pXK-T4 pCR2.1-TOPO containing rfb gene cluster,Amp^(r) this study Kan^(r) pGB2-Sd1 pGB2 containing S. dysenteriaerfb-rfp gene this study cassette, Spc^(r)

Plasmids pGB2 (which is derived from plasmid pSC101) and pCR2.1-TOPO(Invitrogen) were used for cloning and subcloning. Bacterial strainswere grown at 37° C. in Luria-Bertani (LB) broth or on LB agar (Difco).S. enterica serovar Typhi Ty21a strain was grown in SOB (soy broth)medium (Difco). Plasmid-containing strains were selected in mediumcontaining ampicillin (Amp; 100 μg/ml for E. coli and 25 μg/ml for S.enterica serovar Typhi or spectinomycin (Spc; 100 μg for E. coli, 50μg/ml for S. enterica serovar Typhi Ty21a).

PCR and DNA cloning. Unless otherwise noted all DNA manipulations wereperformed essentially by following the procedures outlined by Sambrooket al. (Sambrook, J. et al. 1989 Molecular cloning: a laboratory manual,2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)or by following instructions provided with various commerciallyavailable reagents and kits, including a genomic DNA purification kit,plasmid purification kits and PCR products purification kits (Promega,Madison Wis.). Restriction enzymes (Roche) were used with the suppliedbuffers. Plasmid electroporation was performed with a Gene Pulser(Bio-Rad). All PCR reactions were conducted with ExTaq or LA-Taq (TakaraCo).

Genomic DNA of Shigella dysenteriae serotype 1 strain 1617, isolatedwith a genomic DNA purification kit, was used as a PCR template togenerate the 9.2 kb DNA fragment containing the rfb locus. A 1.6 kb DNAfragment containing the rfp gene was synthesized by PCR from Shigelladysenteriae serotype 1 strain 1617 genomic template material-treated byboiling. The PCR products were used for sequencing studies and forconstruction of the rfb-rfp linked gene region cassette. Sequencingtemplates included PCR products from 1.6 kb to 9.2 kb in Size.

O-Ps expression analyses. Slide agglutination was performed with rabbitantisera against Shigella dysenteriae serotype 1 (B-D Co., Sparks, Md.USA). For immunoblotting, Salmonella, Shigella, and E. coli strains withor without various recombinant plasmids were grown overnight withaeration at 37° C. in LB media containing appropriate antibiotics.Bacteria were pelleted by centrifugation and were lysed in sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) samplebuffer containing 4% 2-mercaptoethanol. The samples were heated at 95°C. for 5 min, and treated with proteinase K for 1 hr, and LPS sampleswere fractionated by 16% Tris-Glycine-SDS-PAGE on a Novex mini-cell gelapparatus (Invitrogen Life Technologies) at 30 mA until tracing dye hadleft the gel. For immunoblotting, LPS bands were transferred topolyvinylidene floride membranes (Schleicher & Schuell, Germany). Themembranes were blocked with 5% nonfat dry milk in Tris-buffered saline(TBS: 20 mM Tris-HCl, 150 mM NaCl, pH 7.5) and were reacted with rabbitpolyclonal antibodies against the O antigen of either Shigelladysenteriae serotype 1 or Salmonella typhi (Difco Laboratories,Michigan, USA), followed by protein A-alkaline phosphatase conjugate.The developing solution consisted of 200 mg of Fast Red TR salt and 100mg of Naphthol NS-MX phosphate (Sigma) in 50 mM Tris buffer, pH 8.0).The silver staining analysis was performed using SilverXpress SilverStaining Kit (Invitrogen) according to the manufacturer's instructions.

DNA sequence and analysis. DNA sequencing was performed with ReadyReactions DyeDeoxy Terminator cycle sequencing kits (Applied Biosystems)and an ABI model 373A automated sequencer. The PCR products includingthe 9.2 kb rfb region and the 1.6 kb rfp gene region, amplified fromgenomic DNA of Shigella dysenteriae serotype 1 strain 1617, were usedfor sequencing and construction of the linked rfb-rfp gene cassette.Sequences were assembled and analyzed by using the Vector NTI suite 9.0software (InforMax, Inc.). DNA homology searches were performed by usingthe Basic Local Alignment Search Tool (BLAST) of National Center forBiotechnology Information. Putative promoters were identified by usingMacVector 6.5 (Accelrys, Burlington, Mass.). The JUMPstart sequence wasfound by using NIH Computational Molecular Biology software, GCG-LeftSequence Comparison Tools, and the JUMPstart sequence identified fromour previous studies at an upstream region of the S. sonnei O antigenlocus (Xu, D. Q. et al. 2002 Infect Immun 70:4414-23). In order toconfirm the fidelity of our sequence data obtained from LA Taq PCRproducts, the Computational Molecular Biology software, GCG-LeftSequence Comparison Tools was also used to compare with homologoussequences from a different Shigella dysenteriae serotype 1 strainprovided by the Sanger Sequencing Institute.

Growth curves and stability of O-antigen expression in the recombinantvaccine strain. Several studies were conducted to determine if theSalmonella vaccine strains carrying a rfb/rfp recombinant expressionplasmid are efficient for growth and stably express the Shiga-1O-antigen. First, growth curves of recombinant strains and controlbacteria under different growth conditions were compared. Shigelladysenteriae serotype 1 O-antigen-specific positive colonies of Sd1-Ty21aand Sd1-E. coli were inoculated into LB broth with or withoutantibiotic. Overnight cultures of each strain were diluted to an OD₆₀₀of approximately 0.1, and growth to the stationary phase was monitored.

Animal immunization study. We are in the process of conducting animalprotection studies to confirm safety and efficacy. In anotherembodiment, we envision removing any antibiotic resistance gene from thefinal plasmid construct and inserting a different selection marker(e.g., a heavy metal ion resistance gene, such as mercury resistancegene) in place of antibiotic resistance to allow for geneticmanipulations. In yet another embodiment, we envision inserting a geneencoding for the Shiga toxin B subunit, which is nontoxic but stimulatesimmunity to whole Shiga toxin, into the final vaccine strain. Thus, inthis embodiment, the final vaccine will trigger antibodies againstShigella dysenteriae serotype 1 LPS and against Shiga toxin to givebetter protection against Shigella dysenteriae serotype 1, and it isenvisioned as providing protection against Shiga toxin-producing E. colistrains to prevent the occurrence of hemolytic uremic syndrome caused byShiga toxin-mediated damage to the kidneys.

Results

Cloning the essential Shigella dysenteriae serotype 1 O-Ps biosyntheticgenes and construction of an O-antigen gene expression cassette.Previous studies showed that the determinants for the production of Oantigen lipopolysaccharide in Shigella dysenteriae serotype 1 aredistributed on the chromosome (i.e., rfb genes) and on a small 9-kbplasmid (FIG. 1). The DNA fragment containing the rfp gene was firstsynthesized by PCR from the whole cell lysate (treated by boiling) ofShigella dysenteriae serotype 1 strain 1617 with the two primers listedbelow and based upon the previously published DNA sequence (GenBankAccession #: M96064): dy5: ttatttccagactccagctgtcattatg (SEQ ID NO: 13);dy6: ccatcgatattggctgggtaaggtcat (SEQ ID NO: 14).

The 1.6 kb PCR fragment was cloned into the pCR 2.1-TOPO cloning vector(Invitrogen). The resulting TOPO-rfp recombinant plasmid, designatedpXK-Tp, was digested with EcoRI, then the EcoRI fragment containing therfp gene was cloned into the EcoRI site of the low copy plasmid pGB2.The resulting pGB2-rfp recombinant plasmid was designated pXK-Bp56.

The large DNA fragment containing the 9.2 kb rfb gene cluster wasamplified from Shigella dysenteriae serotype 1 genomic DNA directly byusing LA Taq polymerase (Takara) cocktail that combines the provenperformance of Taq polymerase with an efficient 3′-5′ exonucleaseactivity for increased proofreading fidelity. The primers used in thisamplification are: SalI-N: cgtatgtcgactgagctctctgaatactctgtcatccagaccaaa(SEQ ID NO: 15) (ref. to GenBank Accession #: AF529080) (a Sailrestriction site is created); BamHI-C:tatcagcttttcactcaactcggcggatccgccctcatac (SEQ ID NO: 16) (ref. toGenBank Accession #: L07293) (a BamHI-C restriction site is created).

Using BLAST, we found that one of four genes which encodes enzymesinvolved in rhamnose biosynthesis of E. coli 026 strain has extensivehomology with a gene (rfbD) of Shigella dysenteriae serotype 1 which haspredicted involvement in rhamnose biosynthesis. In order to identify apotential primer binding site adjacent to the N-terminal region of therfb gene cluster of Shigella dysenteriae serotype 1, a series of primersrecognizing the N-terminal sequence adjacent to the O-antigen genecluster of E. coli 026 were synthesized. We successfully produced a 9.2kb DNA fragment by PCR using a primer (i.e., SalI-N) synthesized fromthe N-terminus of the O-antigen gene cluster of E. coli 026 and anotherprimer (i.e., BamHI-C) synthesized from the previously definedC-terminal region adjacent to the rfb gene cluster of Shigelladysenteriae serotype 1 and using genomic DNA of Shigella dysenteriaeserotype 1 1617 as a template. Previous studies indicated that this 9.2kb DNA fragment contained all essential ORFs of the rfb gene cluster.

The 9.2 kb PCR DNA fragment containing the rfb gene locus was firstcloned into the pCR 2.I-TOPO cloning vector (Invitrogen), resulting inplasmid pXK-T4. In order to combine this rfb gene cluster with thecloned rfp gene, plasmid pXK-T4 was digested with BamHI and SalI, andthe 9.2 kb BamHI-SalI fragment was cloned into plasmid pXK-Bp56, whichhad been cleaved with BamHI and SalI, to produce the linked rfb-rfp geneexpression cassette. The resulting new recombinant low copy pGB-2derivative plasmid was designated pGB2-Sd1 (FIG. 2). As shown in FIG. 2,the rfp gene encoding galactosyl transferase is located downstream ofthe rfb gene cluster and both contain their cognate promoter regions.After pGB2-Sd1 electroporation into E. coli or S. typhi, colonies thatexpress Shigella dysenteriae serotype 1 O-antigen were identified bycolony immunoblotting with Shiga 1-specific antiserum.

Expression of Shigella dysenteriae serotype 1 O-antigen in Salmonellatyphi vaccine strain Ty21a. Plasmid pGB2-Sd1 was transferred byelectroporation into S. enterica serovar Typhi Ty21a. Resultingelectroporants were characterized by colony immunoblot for Shigelladysenteriae serotype 1 O-antigen expression. All colonies showed strongpositive reaction by colony immunoblot screening, and all selected Ty21a(pGB2-Sd1) colonies directed expression of Shiga 1 O-antigen asdetermined by slide agglutination with Shigella dysenteriae serotype1-specific antiserum.

Plasmid-based expression of Shigella dysenteriae serotype 1 O-antigen ineach host was further examined by SDS-PAGE followed by silver stainingand Western immunoblotting with Shigella dysenteriae serotype 1-specificantisera. LPS from wild type Shigella dysenteriae serotype 1 strain 1617gave a typical O-antigen ladder pattern with the predominant chainlength of 17 to 21 O units as detected by both silver stain orimmunoblotting (FIGS. 4A and B).

Silver stain analyses of lipopolysaccharide from various strains (FIG.4A) revealed a series of prominent protein bands that were resistant toprotease K digestion. Despite the presence of these interfering bands,several observations could be made. The control rough E. coli K12carrying the empty pGB2 plasmid vector (lane pGB2.E. coli) as well asthe Shigella dysenteriae serotype 1 60R rough strain (lane Sd1 60R)showed no evidence of LPS ladders, as expected. A faint LPS ladderpattern was seen with the wild type Shigella dysenteriae serotype 1 1617strain (lane Sd1.1617), but was obscured by heavy protein bands in thebottom half of the gel. A similar Shiga 1 ladder pattern was observedmore clearly in the E. coli or Ty21a strains carrying pGB2-Sd1 (lanesSd1.E. coli and Sd1.Ty21a, respectively). S. typhi Ty21a alone showedthe typical repeats of the 9,12 ladder pattern of this serovar (laneTyphi Ty21a).

As shown in FIG. 4B, anti-Shigella dysenteriae serotype 1 O-antigenreactive material was not detected with Shigella dysenteriae serotype 1rough strain 60R (lane Sd1 60R), rough E. coli K-12 carrying pGB-2 (lanepGB-2.E. coli) or S. typhi Ty21a alone (lane Sd1.Ty21a). However,recipient S. typhi Ty21a or E. coli strains carrying pGB2-Sd1 (lanesSd1.Ty21a and Sd1.E. coli) showed typical LPS patterns like that seenwith the Shigella dysenteriae serotype 1 wild type strain (laneSd1.1617).

In this study, the S. enterica serovar Typhi Ty21a-bearing pGB2-Sd1clearly exhibited the typical Shigella dysenteriae serotype 1-specificO-antigen LPS ladder. In contrast to the findings reported earlier, theShigella dysenteriae serotype 1 O-Ps in vaccine strain Ty21a showed acore-linked LPS pattern.

Sequence analysis and a proposed biopathway for Shigella dysenteriaeserotype 1 O-antigen synthesis. A contiguous segment of about 9.2 kb(rfb/rfc region) (GenBank #AY585348) and a 1.6 kb (rfp fragment)(GenBank #AY763519) were sequenced to characterize the Shigelladysenteriae serotype 1 O-antigen biosynthetic genes. Primary analysis ofthe 9.2 kb sequence revealed 9 open reading frames (ORFs); the last openreading frame (orf9) was identified as a small protein coding sequence.In order to demonstrate whether orf9 is essential for Shiga 1 O-antigenbiosynthesis, plasmid pGB2-Sd1 was subjected to digestion with SspBI andBstXI (which are uniquely located in the middle of orf 9), followed byreligation. The new construct, containing a deletion of the middle oforf9, showed identical O-antigen expression compared with the originalplasmid pGB2-Sd1, indicating that orf9 is not involved in O-antigenbiosynthesis.

To confirm the fidelity of the resulting sequence data obtained from PCRproducts synthesized using LA Taq polymerase, our 9.2 kb sequence wascompared with an homologous Shiga 1 rfb region available fromunpublished data using GCG Molecular Comparison Program of the SangerSequencing Center. The results showed 99.98% identity with the Sangersequence from S. dysenteriae strain M131649(M131) and only onenucleotide change (i.e., a G to C transition at position 2450 withinrfbB; accession #: AY585348). In addition, the presumed transcriptionalantiterminator JUMPstart sequence:cagtggctctggtagctgtaaagccaggggcggtagcgt (SEQ ID NO: 17) was identifiedat by 643-680 (GenBank accession#:AY585348) of the amplified rfb regionof Shiga 1 strain 1617.

The Shigella dysenteriae serotype 1 O antigen genes. The properties ofthe nine essential genes including eight ORFs from the rfb locus plusthe rfp gene, summarized in Table 2, were obtained from homologysearches. The putative genes involved in biosynthesis of thetetrasaccharide repeating unit: L-Rhap, L-Rhap, D-Galp, and D-GlcNAcp aswell as genes for a unit processing (e.g., encoding O antigentransporter/flipase and polymerase) were identified. The genes involvedin the rhamnose biopathway, rfbB, rfbC, rfbA and rfbD, (Klena, J. D. etal. 1993 Molec Microbio 19:393-402) share 98.5, 99, 99, and 93%identity, respectively, with the rhamnose biosynthetic genes rmlB, rmlD,rmlA and rmlC of E. coli 026. The enzymatic working order of the fourproteins in this pathway are: RfbA, RfbB, RfbC and RfbD. RfbA/RmlA is aglucose-1-phosphatate thymidylytransferase, which links Glu-1-P to acarrier nucleotide creating dTDP-glucose for further chemicaltransformation. RfbB/RmlB is an dTDP-D-glucose 4,6-dehydratase, whichcatalyzes the second step in the rhamnose biosynthetic pathway: thedehydration of dTDP-D-glucose to form dTDP-4-keto 6-deoxy-D-glucose.RfbC/RmlC is dTDP-4-dyhydrorhamnose reductase. RfbD/RmlD is adTDP-4-dehydrorhamnose 3,5-epimerase, which catalyses the terminalreaction in dTDP-L-rhamnose biosynthesis, reducing the C4-keto group ofdTDP-L-lyxo-6-deoxy-4-hexulose to a hydroxyl resulting in the productdTDP-L-rhamnose. RfbX is putative O antigen transporter, which belongsto the Wzx gene family involved in the export of O antigen and teichoicacid. This protein shows only 53% identity to that of E. coli K-12. Thenext Orf is rfc, which was a member of the Wzy protein family of Oantigen polymerases. Wzy proteins usually have several transmembranesegments and a large periplasmic loop which interacts with the O antigenchain length determinant Cld/wzz to control O-Ps repeat unit chainlength and distribution on the cell surface. There are two putativerhamnosyltransferases which are located at the end of this rfb locus.The transferase must recognize both the sugar nucleotide and therecipient polymer to which the sugar is transferred, forming a specificglycosidic linkage. There are two rhamnosyltranferases which work intandem to link the 2 rhamnoses at the end of the O-repeat unit. Wesuggest that the S. typhi chromosomal Rfe, which is very conserved ingram-negative bacteria, is a GlcNac transferase which first adds GlcNActo the ACL (antigen carrier lipid/acyl lipid carrier/undecaprenolphosphate). Rfp is a galactosyl transferase, which normally transfersthe Gal moiety from UDP-Gal to the GluNAc-bound ACL. Following these twosugars, the 2 terminal rhamnoses are transferred to complete thetetrasaccharide O-repeat unit.

Summary

The O-Ps biosynthetic determinants from Shigella dysenteriae serotype 1strain 1617 were cloned from both the chromosome (i.e., rfb locus) and asmall 9 kb plasmid (i.e., the rfp gene). The separate rfb locus and ifpregion covering ˜11 kb total DNA were sequenced entirely. Sequencingdata and genetic deletion studies in one terminal orf revealed that 8Rib orf's and the single Rfp orf are necessary for O-Ps biosynthesis. Alow copy pGB2 vector containing both the rfb and rfp loci in tandemlinkage with their cognate promoters was constructed (i.e., pGB2-Sd1).This plasmid is genetically stable and promotes the expression ofShigella dysenteriae serotype 1 O-Ps antigen as a typical core-linkedstructure in both E. coli and S. Typhi recipients. Sequence comparisonsrevealed proposed gene functions for the 9 required Orf's that result inthe biosynthesis of a tetrasaccharide repeat O-unit as well 9*as itsprocessing, transport and linkage to core oligosaccharide. We anticipatethat use of this cloned antigen locus in a live, attenuated Salmonelladelivery system will lead to a safe, oral vaccine for protection againstthis severe form of shigellosis.

While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All figures, tables, andappendices, as well as patents, applications, and publications, referredto above, are hereby incorporated by reference.

The invention claimed is:
 1. An attenuated strain of Salmonellacomprising core-linked Shigella dysenteriae serotype 1 O-specificpolysaccharide (O-Ps) and DNA encoding O antigen biosynthesis, said DNAselected from the group consisting of: a) the DNA sequence set out inany one of SEQ ID NOs: 1 and 2 and nucleic acid molecules from thespecies Shigella dysenteriae serotype 1 that share at least about 90%sequence identity with the nucleic acid molecule of SEQ ID NO: 1 or 2;b) DNA encoding Shigella dysenteriae serotype 1 polypeptides encoded byany one of SEQ ID NOs: 1 and 2 and nucleic acid molecules from thespecies Shigella dysenteriae serotype 1 that share at least about 90%sequence identity with the nucleic acid molecule of SEQ ID NO: 1 or 2;and c) DNA encoding variants of Shigella dysenteriae serotype 1biosynthesis polypeptides encoded by any one of SEQ ID NOs: 1 and 2,wherein each variant comprises O antigen biosynthesis polypeptide, andwherein one or more of the specified amino acids is deleted or replaced,or wherein one or more non-specified amino acids are added without lossof Shigella dysenteriae serotype 1 O antigen or protective immunologicalactivity of the O antigen biosynthesis gene product.
 2. The attenuatedstrain of claim 1, said DNA being present on a plasmid.
 3. Theattenuated strain of claim 1, said DNA being under the control of itscognate promoters.
 4. A composition of matter comprising the attenuatedstrain of claim 1 in combination with a physiologically acceptablecarrier.
 5. A method of prophylactic or therapeutic treatment ofbacterial infection comprising administering a prophylactically ortherapeutically effective amount of the attenuated strain of claim 1 toan individual for prescription of said treatment.
 6. A method of makinga vaccine comprising combining the attenuated strain of claim 1 with aphysiologically acceptable carrier.
 7. The attenuated strain of claim 1,wherein the Salmonella is selected from the group consisting ofSalmonella typhimurium and Salmonella typhi.
 8. An attenuated strain ofShigella comprising core-linked Shigella dysenteriae serotype 1O-specific polysaccharide (O-Ps) and DNA encoding O antigenbiosynthesis, said DNA selected from the group consisting of: a) the DNAsequence set out in any one of SEQ ID NOs: 1 and 2 and nucleic acidmolecules from the species Shigella dysenteriae serotype 1 that share atleast about 90% sequence identity with the nucleic acid molecule of SEQID NO: 1 or 2; b) DNA encoding Shigella dysenteriae serotype 1polypeptides encoded by any one of SEQ ID NOs: 1 and 2 and nucleic acidmolecules from the species Shigella dysenteriae serotype 1 that share atleast about 90% sequence identity with the nucleic acid molecule of SEQID NO: 1 or 2; and c) DNA encoding variants of Shigella dysenteriaeserotype 1 biosynthesis polypeptides encoded by any one of SEQ ID NOs: 1and 2, wherein each variant comprises O antigen biosynthesispolypeptide, and wherein one or more of the specified amino acids isdeleted or replaced, or wherein one or more non-specified amino acidsare added without loss of Shigella dysenteriae serotype 1 O antigen orprotective immunological activity of the O antigen biosynthesis geneproduct.
 9. The attenuated strain of claim 8, wherein the Shigella isselected from the group consisting of Shigella flexneri and Shigellasonnei.
 10. The attenuated strain of claim 8, said DNA being present ona plasmid.
 11. The attenuated strain of claim 8, said DNA being underthe control of its cognate promoter.
 12. A composition of mattercomprising the attenuated strain of claim 8 in combination with aphysiologically acceptable carrier.
 13. A method of prophylactic ortherapeutic treatment of bacterial infection comprising administering aprophylactically or therapeutically effective amount of the attenuatedstrain of claim 8 to an individual for prescription of said treatment.14. A method of making a vaccine comprising combining the attenuatedstrain of claim 8 with a physiologically acceptable carrier.